Abstract

Developmental signaling pathways often activate their own inhibitors. Such inhibitory feedback has been suggested to restrict the spatial and temporal extent of signaling or mitigate signaling fluctuations, but these models are difficult to rigorously test. Here, we determine whether the ability of the mesendoderm inducer Nodal to activate its inhibitor Lefty is required for development. We find that zebrafish lefty mutants exhibit excess Nodal signaling and increased specification of mesendoderm, resulting in embryonic lethality. Strikingly, development can be fully restored without feedback: Lethal patterning defects in lefty mutants can be rescued by ectopic expression of lefty far from its normal expression domain or by spatially and temporally uniform exposure to a Nodal inhibitor drug. While drug-treated mutants are less tolerant of mild perturbations to Nodal signaling levels than wild type embryos, they can develop into healthy adults. These results indicate that patterning without inhibitory feedback is functional but fragile.

eLife digest

During animal development, a single fertilized cell gives rise to different tissues and organs. This ‘patterning’ process depends on signaling molecules that instruct cells in different positions in the embryo to acquire different identities. To avoid mistakes during patterning, each cell must receive the correct amount of signal at the appropriate time.

In a process called ‘inhibitory feedback’, a signaling molecule instructs cells to produce molecules that block its own signaling. Although inhibitory feedback is widely used during patterning in organisms ranging from sea urchins to mammals, its exact purpose is often not clear. In part this is because feedback is challenging to experimentally manipulate. Removing the inhibitor disrupts feedback, but also increases signaling. Since the effects of broken feedback and increased signaling are intertwined, any resulting developmental defects do not provide information about what feedback specifically does. In order to examine the role of feedback, it is therefore necessary to disconnect the production of the inhibitor from the signaling process.

In developing embryos, a well-known signaling molecule called Nodal instructs cells to become specific types – for example, a heart or gut cell. Nodal also promotes the production of its inhibitor, Lefty. To understand how this feedback system works, Rogers, Lord et al. first removed Lefty from zebrafish embryos. These embryos had excessive levels of Nodal signaling, did not develop correctly, and could not survive. Bathing the embryos in a drug that inhibits Nodal reduced excess signaling and allowed them to develop successfully. In these drug-treated embryos, inhibitor production is disconnected from the signaling process, allowing the role of feedback to be examined. Drug-treated embryos were less able to tolerate fluctuations in Nodal signaling than normal zebrafish embryos, which could compensate for such disturbances by adjusting Lefty levels.

Overall, it appears that inhibitory feedback in this patterning system is important to compensate for alterations in Nodal signaling, but is not essential for development. Understanding the role of inhibitory feedback will be useful for efforts to grow tissues and organs in the laboratory for clinical use. The results presented by Rogers, Lord et al. also suggest the possibility that drug treatments could be developed to help correct birth defects in the womb.

Lefty production is coupled to Nodal signaling, forming a negative feedback loop that is conserved from sea urchins to humans. For example, in zebrafish, lefty1 and lefty2 are induced by endogenous Nodal signaling at the blastoderm margin, expression of Nodal can drive ectopic lefty production, and loss of Nodal signaling abolishes expression of lefty (Figure 1A,B) (Meno et al., 1999). Despite the ubiquity of this motif, the functions provided by coupling Nodal activation to inhibition remain unclear.

Several roles for Lefty feedback have been suggested. First, Nodal and Lefty were proposed to form a reaction-diffusion patterning system that regulates both mesendoderm formation and left/right patterning in zebrafish (Chen and Schier, 2002; Kondo and Miura, 2010; Meinhardt, 2009; Müller et al., 2012; Schier, 2009; Shen, 2007; Shiratori and Hamada, 2006). Notably, Nodal and Lefty fulfill the key biophysical requirements of a classical reaction-diffusion system: Both Nodal ligands (Cyclops and Squint) act as short-range (Cyclops) and mid-range (Squint) activators that induce their own expression as well as that of Lefty1 and Lefty2, which act as long-range, highly mobile inhibitors (Chen and Schier, 2002; 2001; Feldman et al., 2002; Meno et al., 1999; Müller et al., 2012). Second, Lefty was argued to temporally restrict Nodal signaling by creating a ‘window’ of signaling competence (van Boxtel et al., 2015). In this model, Nodal signaling proceeds until sufficient Lefty accumulates to shut down further signaling. Third, theoretical studies suggest that inhibitory feedback has the potential to mitigate fluctuations in signaling (Lander et al., 2009). Deleterious increases or decreases in Nodal signaling could therefore be offset by adjustments in Lefty-mediated inhibition, ensuring robust development in the face of variation in the external environment or expression of pathway components.

To understand the role of inhibitory feedback in the Nodal/Lefty patterning system, we created embryos in which Nodal inhibition was decoupled from Nodal signaling. We found that inhibitory feedback mitigates signaling perturbations but is dispensable for development.

Mutations in nodal genes partially rescue lefty1-/-;lefty2-/- mutants

The patterning defects in lefty double mutants show a requirement for reduction of Nodal activity but do not establish a requirement specifically for inhibitory feedback. It is possible that a reduction in Nodal signaling by means other than inhibitory feedback could support patterning. For example, reducing nodal gene dosage could suppress lefty double mutant defects (Chen and Schier, 2002; Feldman et al., 2002; Meno et al., 1999). In support of this hypothesis, mutations in the Nodal genes squint or cyclops suppressed multiple aspects of the lefty1-/-;lefty2-/- mutant phenotype (Figure 4). cyclops-/-;lefty1-/-;lefty2-/- (Figure 4C’’) and squint-/-;lefty1-/-;lefty2-/- (Figure 4F’’) mutants formed eyes and full-length tails, structures missing in lefty1-/-;lefty2-/- mutants (Figure 4A’’, D’’, M,N). Moreover, upregulation of mesendodermal gene expression was suppressed in squint-/-;lefty1-/-;lefty2-/- mutants compared to lefty1-/-;lefty2-/- mutants (Figure 4G–L’’’). Although these triple mutants are not viable, two functional nodal alleles are sufficient to generate mesendodermal gene expression patterns that are similar to those observed in wild type embryos with four nodal alleles and four lefty alleles. In previous studies, removal of squint, but not cyclops, partially suppressed defects in lefty double morphants (Chen and Schier, 2002; Feldman et al., 2002), but our results indicate that Lefty inhibits both Squint and Cyclops. The failure to fully rescue development may reflect an inability to precisely modulate Nodal dosage with this genetic approach, but reduction of Nodal dosage can partially rescue development in the complete absence of Lefty-mediated inhibition.

nodal mutations suppress lefty1-/-;lefty2-/- mutant defects.

(A–F’’) Lateral views of embryos with indicated genotypes at 1 day post-fertilization (dpf). Embryos are progeny from an incross of cycm294/+;lft1-/-;lft2+/- (A–C’’) or sqtcz35/+;lft1-/-;lft2+/- (D–F’’) adults, and were genotyped after imaging (see Materials and methods for details). In contrast to lft1-/-;lft2-/- mutants (A’’, D’’), nodal+/-;lft1-/-;lft2-/- mutants (B’’, E’’) have long tails and well-defined heads, and some nodal-/-;lft1-/-;lft2-/- mutants (C’’,F’’) have eyes in addition to full-length tails. nodal-/-;lft1-/-;lft2-/- and nodal+/-;lft1-/-;lft2-/- mutants are not viable. cyc homozygotes exhibit the expected curved body axis (Sampath et al., 1998) (C,C’), but curvature is reduced in cyc-/-;lft1-/-;lft2-/- embryos (C’’). (G–L’’’) In situ hybridization assessing expression of endodermal (sox32/casanova) (G–I’’’) or mesodermal (noto/floating head) (J–L’’’) genes in the indicated genotypes (dorsal views). Mesendoderm upregulation is less pronounced in sqt-/-;lft1-/-;lft2-/- compared to lft1-/-;lft2-/- mutants. (M–N) 72 embryos from a cyc+/-;lft1-/-;lft2+/- incross (M) and 84 embryos from a sqt+/-;lft1-/-;lft2+/- incross (N) were scored and imaged at 1 dpf, and subsequently genotyped. Number of embryos of each genotype with the indicated phenotype at 1 dpf is shown. Together, these results demonstrate that loss of sqt or cyc can suppress lft1-/-;lft2-/- mutant phenotypes.

Discussion

The results in this study show that inhibitory feedback in the Nodal/Lefty system stabilizes Nodal signaling but is not essential for mesendoderm patterning and viability. The rescue of lefty mutants by ectopic lefty expression (Figure 5, Figure 5—figure supplements 1 and 2) and exposure to Nodal inhibitor drug (Figure 6, Figure 6—figure supplements 1–4, Figure 2—figure supplement 1) is consistent with the high diffusivity of Lefty measured previously (Müller et al., 2012), but is surprising in light of the functions assigned to inhibitory feedback. Specifically, inhibitory feedback has been implicated in (1) shutting down pathway activity at the appropriate time to generate a pulse or window of signaling, (2) shaping spatial signaling profiles, (3) acting as part of self-organizing reaction-diffusion systems, and (4) mitigating fluctuations in signaling activity. Below, we discuss our results in the context of these models.

First, inhibitory feedback has been suggested to turn off signaling activity when it is no longer needed (Dessaud et al., 2007; Freeman, 2000; Golembo et al., 1996; Ribes and Briscoe, 2009; Shiratori and Hamada, 2006). For Nodal-mediated patterning, it has been proposed that progressively increasing Lefty levels shut down Nodal signaling at the onset of gastrulation (van Boxtel et al., 2015). However, we find that Nodal signaling is already increased in lefty double mutants by sphere stage, suggesting an earlier role for Lefty (Figure 2). Moreover, Lefty can be replaced by an inhibitor drug added as early as the 8 cell stage (Figure 6, Figure 6—figure supplements 1–4, Figure 2—figure supplement 1), demonstrating that mesendoderm patterning can proceed without progressively increasing inhibition and without temporally precise feedback inhibition. Our results do not rule out that Lefty accumulation shuts down Nodal signaling during normal development, but they do indicate that this is not an absolute requirement for patterning.

Third, the dispensability of inhibitory feedback in the Nodal/Lefty system raises questions about the role of Nodal and Lefty as an activator/inhibitor pair in self-organizing reaction-diffusion models. Although Nodal and Lefty fulfill the key regulatory and biophysical requirements of a short-to-mid-range autocatalytic activator and a long-range feedback inhibitor (Hamada, 2012; Meno et al., 1999; Müller et al., 2012; Schier, 2009), our finding that development can be normal without inhibitory feedback indicates that this system does not require the ability to form self-organizing reaction-diffusion patterns. Instead, it is conceivable that the pre-patterning of the early embryo by maternal factors and the local activation of Nodal eliminate the need for self-organizing pattern generation by the Nodal/Lefty circuit. In this scenario, Nodal and Lefty may have constituted a reaction-diffusion activator/inhibitor pair in ancestral organisms but, through the addition of other regulatory layers, mesendoderm patterning lost the requirement for inhibitory feedback.

Finally, feedback inhibition has been implicated in buffering fluctuations in pathway activity (Barkai and Shilo, 2009; Eldar et al., 2003; Lander et al., 2009). Feedback may be required to optimize inhibitor levels, as suggested by the narrow range (~2 fold) of inhibitor concentrations that rescue Lefty loss (Figure 6I, Figure 6—figure supplements 1 and 2). Indeed, the adjustment of lefty expression in response to slight alterations in Nodal signaling (Figure 7) and the failure of feedback-decoupled embryos to cope with perturbations in Nodal signaling (Figure 8) support this idea. The ability to dynamically adjust pathway activity may allow the embryo to create reliable patterns in the face of endogenous signaling fluctuations and uncertain environmental conditions. We note, however, that Lefty feedback does not protect the embryo against all perturbations: Drug-rescued lefty mutants actually fared better than wild type embryos when challenged with injection of squint mRNA (Figure 8—figure supplement 1). Dissecting why Lefty feedback corrects some perturbations but not others will provide a window into the mechanisms and limits of robust patterning.

Our results have implications not only for the roles of feedback inhibition during development, but also demonstrate the feasibility of preventing patterning defects with small molecule drug exposure. Although suggested applications to human embryos might currently seem fanciful and would be challenging and fraught with ethical concerns, embryos bearing compromised patterning circuits could be identified by sequencing a single embryonic cell, and birth defects could be prevented by exposure to the appropriate small molecule. More generally, our study adds a new facet to recent revisions of classical patterning models (Alexandre et al., 2014; Chen et al., 2012; Dominici et al., 2017; Dubrulle et al., 2015; Varadarajan et al., 2017). For example, a tethered form of Wingless can replace endogenous Wingless, challenging models in which a gradient of diffusing Wingless is indispensable for tissue patterning (Alexandre et al., 2014). In the same vein, our observations challenge models in which inhibitory feedback is an absolute requirement for patterning and viability, but support the idea that inhibitory feedback enhances robustness by stabilizing signaling during development.

Embryos from an incross of the TLAB wild type strain were injected at the one-cell stage with 300–450 pg mRNA encoding each TALEN pair. Injected fish were grown using standard fish husbandry protocols and fin clipped as adults. Genomic DNA was generated from fin material using the Hot Shot method (Meeker et al., 2007). To identify animals carrying mutations, PCR using primers flanking the target sites was carried out and the resulting amplicons were re-annealed and digested with mismatch-cleaving T7 endonuclease I (NEB) (Mussolino et al., 2011). PCR products from positive animals were cloned using a TOPO TA kit (Life Technologies) and sequenced. Positive animals were outcrossed and progeny were sequenced and tested for germline transmission.

Primers flanking lefty TALEN target sites:

lefty1 forward primer: catgtatcaccttccctctgatgtc

lefty1 reverse primer: gcattagcctatatgttaacttgcac

lefty2 forward primer: tacttatcaacatgagcatcaatgg

lefty2 reverse primer: gaattgtgcataagtaacccacctg

Genotyping

lefty1: The 13-base-pair deletion in lefty1a145 destroys a PshAI restriction site. To genotype the lefty1 locus, PCR amplicons were generated using primers flanking the deletion and subsequently digested with PshAI endonuclease (NEB). Genotyping primers were identical to the lefty1 forward/reverse primers described above. Complete digestion by PshAI indicates that both alleles are wild type, partial digestion indicates heterozygosity, and failure to digest indicates homozygosity for the lefty1a145 mutation.

lefty2: The 11-base-pair deletion in lefty2a146 was detected using a mutant-specific forward primer that spans the deletion. A forward primer specific to the wild type allele was also designed, as well as a reverse primer that is fully complementary to both alleles. To genotype the lefty2 locus, PCR was carried out using either the wild type- or mutant-specific forward primer and the common reverse primer. A band with the wild type- but not mutant-specific primer indicates that both alleles are wild type, bands with both primer sets indicate heterozygosity, and a band with the mutant- but not wild type-specific primers indicates homozygosity for the lefty2a146 mutation. Optimal PCR conditions: Taq polymerase, 25 cycles, 57°C annealing temperature.

lefty2 wild type genotyping forward primer: cattttgaccacagcgat

lefty2 mutant genotyping forward primer: gttcattttgaccactcac

The common reverse primer was identical to lefty2 reverse primer described above.

squint: The squintcz35 allele has a ~ 1.9 kb insertion in exon 1, and was detected as in (Feldman et al., 1998).

cyclops: The cyclopsm294 mutation destroys an AgeI restriction site, and was detected as in (Sampath et al., 1998).

lefty and squint expression constructs

To generate mRNA from all constructs, plasmids were linearized with NotI-HF endonuclease (NEB) and purified using a Qiagen PCR clean-up kit. Capped mRNA was generated from linearized plasmid using an SP6 mMessage mMachine kit (Ambion) and purified with a Qiagen RNeasy kit.

After purification, mRNA was quantified using a NanoDrop spectrophotometer (ThermoFisher) and diluted to the appropriate concentration. For microinjections, a micrometer was used to adjust the drop volume to 0.5 nl. Depending on the concentration of the injection mix, a total volume of 1–2 nl was injected per embryo.

Lefty1-GFP, Lefty2-GFP, and untagged Lefty1 and Lefty2 constructs used in Figures 5 and 7 were identical to those in (Müller et al., 2012). These constructs lack endogenous UTRs and contain the consensus Kozak sequence gccacc immediately preceding the start codon.

The 13 bp lefty1a145 mutation removes part of the endogenous Kozak sequence (gaaaag). Therefore, lefty1 constructs containing this endogenous Kozak were generated, rather than the consensus Kozak sequence gccacc as in (Müller et al., 2012). Primers with either the endogenous Kozak sequence (for the wild type construct) or the truncated endogenous sequence and deleted region of coding sequence (for the mutant construct) were designed, and the Lefty1 construct from (Müller et al., 2012) was used as a PCR template. The resulting fragments were cloned into BamHI and XhoI sites in pCS2(+). Both constructs lack endogenous UTRs.

The lefty2 wild type construct was the same used in (Müller et al., 2012) and in the transplantation experiments in Figure 5. The lefty2a146 construct was made by generating cDNA from lefty2 homozygous embryos, amplifying the mutant lefty2 coding sequence, and cloning the resulting fragment into ClaI and XhoI in the pCS2(+) vector. In addition to the 11 bp deletion, the mutant construct contains three silent SNPs at position 184 (T->C), 932 (A->C), and 943 (T->A). Both constructs lack endogenous UTRs and contain the consensus Kozak sequence gccacc immediately preceding the start codon.

Nodal overexpression experiment: The construct used to generate squint mRNA in the Nodal overexpression experiment (Figure 8—figure supplement 1) was identical to that used in (Müller et al., 2012). This construct lacks endogenous UTRs and contains the consensus Kozak sequence gccacc immediately preceding the start codon.

α-pSmad2 immunofluorescence

The protocol was modified from (van Boxtel et al., 2015). Briefly, embryos were fixed in 4% formaldehyde (in 1x PBS) overnight at 4°C, washed in PBST (1x PBS + 0.1% (w/v) Tween 20), manually deyolked, dehydrated in a MeOH/PBST series (25%, 50%, 75%, and 100% MeOH), and stored at −20°C until staining. To prepare for staining, embryos were rehydrated in a MeOH/PBSTr (1x PBS + 1% (w/v) Triton X-100) series (75%, 50%, and 25% MeOH), washed 3x in PBSTr, and incubated for 20 min in ice-cold acetone. Embryos were then washed 3x in PBSTr, incubated in antibody binding buffer (PBSTr +1% (v/v) DMSO) for two hours at room temperature, then incubated overnight at 4°C with a 1:1000 dilution of α-pSmad2 antibody (Cell Signaling Technology #8828, Danvers, MA, USA) in antibody binding buffer. After primary treatment, embryos were washed 6x in PBSTr, incubated in antibody binding buffer for 30 min at room temperature, and incubated for two hours at room temperature with a 1:2000 dilution of goat α-rabbit Alexa 647 conjugate (ThermoFisher A-21245) in PBSTr +1% (v/v) DMSO. Embryos were then washed 6x in PBSTr, 3x in PBS and incubated with 200 nM Sytox green in PBS for 30 min at room temperature. Finally, embryos were washed 3x in PBS and dehydrated in a MeOH/PBS series (50% and 100% MeOH). Stained embryos were stored at −20°C in 100% MeOH until imaging.

α-pSmad2 imaging

Embryos were mounted in agarose and cleared with 2:1 benzyl benzoate:benzyl alcohol (BBBA) (Yokomizo et al., 2012). Briefly, a dehydrated embryo was dropped into molten low-melting point agarose (1% (w/v) in H2O), transferred onto a coverglass and oriented manually. All embryos (other than sphere stage) were mounted ‘margin down’ (i.e. with the animal/vegetal axis parallel to the coverglass, see below for rationale). Shield stage embryos were rolled to ensure that the dorsal/ventral axis was parallel to the coverglass. Sphere stage embryos were mounted with the animal/vegetal axis perpendicular to the coverglass (animal pole facing up). The agarose drop was then dehydrated with three washes of 100% MeOH, two washes of 50:50 (v/v) BBBA:MeOH, and 3 washes of BBBA. Cleared embryos were then sealed to a microscope slide using fast wells reagent reservoirs (Grace Bio-Labs). Imaging was performed on Sytox Green and Alexa 647 channels using an LSM 700 confocal microscope (20x air objective, 0.5 NA). Image stacks extended from the embryo margin (adjacent to the coverglass) to beyond the center of the embryo. Z-planes were spaced at 2 μm intervals.

α-pSmad2 image segmentation and quantification

Quantification of pSmad2 and Sytox green staining intensity in laterally-mounted embryos was performed using the ten z-slices surrounding the center of the embryo axis (i.e. five slices above and five slices below the embryo center). This region was chosen to minimize artifacts due to light scattering, which causes a decrease in apparent fluorescence intensity in deeper tissue planes. Our procedure—looking only at slices close to the ‘central plane’ of the embryo—allows the entire gradient to be sampled within each slice, and ensured that all data were taken from planes within a narrow range of imaging depths, effectively controlling for signal drop-off with imaging depth.

Nuclei were segmented from the Sytox Green channel images using a custom pipeline implemented in MATLAB (Source code 1). Briefly, out-of-plane background signal was approximated by blurring adjacent z-slices (i.e. the slice above and below the plane being segmented) with a Gaussian smoothing kernel and summing. This background was subtracted from the segmentation image, and preliminary boundaries for nuclei were identified by adaptive thresholding (http://homepages.inf.ed.ac.uk/rbf/HIPR2/adpthrsh.htm) of the resulting image. Spurious objects were discarded by morphological filtering (based on object size). Final segmentation boundaries were defined after manual checking and correction with a custom MATLAB script (Source code 1). The fluorescence intensity of each segmented nucleus was defined as the mean intensity of its constituent pixels.

The distance of each nucleus from the margin was defined along a curved embryo contour (Figure 2—figure supplement 1). This contour was defined by 1) projecting all segmented nuclei centroids onto a single z-plane, 2) creating a full embryo ‘mask’ by filling a convex hull containing all of these points, 3) identifying the left and right margin boundaries as the points of maximum curvature on the convex hull, 4) taking the distance transform of the embryo mask, and 5) stepping along the ‘valley’ of the distance transform that connects the left and right margins (as defined above). This rough contour was then smoothed using a Savitsky-Golay filter to yield the final contour. The position of each nucleus was then projected onto the contour, and the distance from the margin (as plotted in Figure 2C and Figure 2—figure supplement 1) was determined as the distance to the closest margin along this curve.

In situ hybridization

Embryos were fixed overnight at 4°C with 4% formaldehyde in PBS. In situ hybridization was carried out as in (Thisse and Thisse, 2008) and representative embryos were imaged in 2:1 benzyl benzoate:benzyl alcohol (BBBA) with a Zeiss Axio Imager.Z1 microscope. When genotyping was necessary, genomic DNA was generated after imaging as in (Meeker et al., 2007) and used in the described genotyping assays.

Decision letter

Deborah Yelon

Reviewing Editor; University of California, San Diego, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Nodal patterning without Lefty inhibitory feedback is functional but fragile" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this manuscript, Rogers et al. devise an interesting test of the relevance of negative feedback in a signaling circuit during early zebrafish development. Nodal signaling, which is activated by the ligands Cyclops and Squint, is kept under control by two secreted feedback inhibitors, Lefty 1 and Lefty 2. Here, the authors investigate the relevance of this regulatory interaction by assessing the effect of exogenous Nodal inhibitors in embryos lacking Lefty 1/2. One of their most remarkable results is that these mutants can be fully rescued by addition of a chemical inhibitor of Nodal signalling (at the just-right dose, but in a non-spatially restricted manner). This result clearly shows that signaling-dependent feedback inhibition is not required for normal development under laboratory conditions. The Lefty proteins are not needed to shut down Nodal signaling through increased production, nor are they absolutely essential for mesendodermal patterning that is compatible with viability. The elegance of the reaction-diffusion model also seems to be dispensable under these circumstances. The authors then suggest that feedback inhibition may nevertheless be important to buffer against fluctuations of signaling activity. The latter argument makes sense but is incompletely investigated; some of the suggested revisions may help to strengthen this argument.

Overall, this is a very strong and well-written manuscript, leading to a reconsideration of the concept of feedback loops as exemplified by the roles of Nodal ligands and their induced inhibitors Lefty 1/2. This work will undoubtedly generate a lot of discussion in the field. The large amount of data and the careful performance of the experiments will be greatly appreciated by those who want to carefully evaluate the data in light of various models for pattern formation. Consideration of the issues listed below is likely to strengthen the work and enhance its impact.

Essential revisions:

1) The authors suggest that there is a sweet spot of signaling activity that is required for normal development. This is defined by the dose of inhibitor that is required to support development of the double mutant. An alternative (quantitative) measure of signaling activity (e.g. western blot with anti-pSMAD) in chemically and genetically rescued embryos would strengthen the authors' argument. This would allow them to estimate the tolerance to variation in signaling activity, which is relevant to their argument that the normal function of feedback is to ensure reliable signaling at the right level.

2) The authors predict that signaling activity would be variable in the absence of feedback. Is it possible to strengthen this argument by measuring embryo to embryo variation among a population of drug-rescued Lefty double mutants (no feedback) with a quantitative measure as suggested above? Would such variation fall outside the tolerance measured in the previous experiment?

3) One possible extension of the finding described is that the spatial requirement for Nodal signalling is not very stringent. Have the authors attempted a converse rescue experiment, i.e. to activate Nodal signaling uniformly, either with a drug or RNA injection in a cyclops;squint double mutant. Would they expect a just-right level of global signaling that rescues development?

4) Regarding the statement "Thus, an ectopic, Nodal-independent source of Lefty at the animal pole can replace endogenous, Nodal-induced Lefty at the margin": Is this confirmed by fate-mapping (with Alexa 488-dextran) of the transplanted, Lefty-expressing cells? Figure 5E, F shows the position of cells shortly after transplantation, but it is quite possible that the transplanted cells move down into the margin during epiboly/gastrulation, providing a more localized source of exogenous Lefty.

5) It is somewhat surprising that there is not a graded effect of loss of lefty alleles, i.e. that the embryonic phenotypes and response readouts (in situs and pSmad2) do not show a graded response, and that loss of one, two or three alleles of Lefty1/2 seem to be equivalent. Is it correct that there is no dose response? What are the phenotypes of the genetic combinations with just one functional allele?

6) In the Discussion, "While Lefty is not essential for viability per se" is an overstatement. The lefty double mutants are not viable. Although they can be experimentally rescued by drug or resupply of Lefty, this does not alter the requirement for functional Lefty during development (whether provided by expression or downstream pathway inhibition), as demonstrated by the double mutant phenotype. It is important in the interpretation of the results and the discussion of the results to uncouple the concept of dynamic feedback inhibition with the concept of the requirement of Lefty to be in the game, at least to modulate Nodal activity levels for viability.

Author response

Essential revisions:

1) The authors suggest that there is a sweet spot of signaling activity that is required for normal development. This is defined by the dose of inhibitor that is required to support development of the double mutant. An alternative (quantitative) measure of signaling activity (e.g. western blot with anti-pSMAD) in chemically and genetically rescued embryos would strengthen the authors' argument. This would allow them to estimate the tolerance to variation in signaling activity, which is relevant to their argument that the normal function of feedback is to ensure reliable signaling at the right level.

We thank the reviewers for this helpful suggestion. We have performed additional experiments to address this point: To examine the tolerance to variation in signaling activity, we used pSmad2 immunofluorescence to measure Nodal signaling in lft double mutants exposed to rescuing, sub-rescuing, and excessive doses of inhibitor drug (new Figure 6—figure supplement 4). Mutants exposed to sub-rescuing doses had expanded Nodal signaling gradients compared to rescued mutants. In contrast, mutants exposed to excessive doses exhibited pSmad2 gradients with diminished amplitude. Concurrently, treatment with excessive or insufficient Nodal inhibitor doses resulted in morphological phenotypes consistent with partial Nodal loss of function and gain of function, respectively. Our new results therefore suggest upper and lower bounds of tolerance to signaling alterations, and support the argument that mechanisms to maintain signaling within these bounds are important for patterning. We have updated the main text accordingly.

2) The authors predict that signaling activity would be variable in the absence of feedback. Is it possible to strengthen this argument by measuring embryo to embryo variation among a population of drug-rescued Lefty double mutants (no feedback) with a quantitative measure as suggested above? Would such variation fall outside the tolerance measured in the previous experiment?

We thank the reviewers for raising this possibility. We have performed additional experiments to address this point: Using pSmad2 immunofluorescence, we observed similar levels of signaling gradient variability in wild type and drug-rescued lft mutants (Author Response Image 1). This result provides further support for the remarkable ability of the inhibitory drug to rescue lft mutants to a wild type phenotype. Although this result does not provide additional evidence for a role of inhibitory feedback in reducing variation, there are technical limitations to the sensitivity of this experiment using currently available methods. To control for technical variability in staining, we followed the accepted practice of normalizing each embryo’s gradient to its total intensity. This approach is well suited to analyze variability in gradient shape but it normalizes biological variability in gradient amplitude. It is therefore conceivable that there might be differences in amplitude variability between wild type and rescued mutants. In future studies we will attempt to develop more quantitative measures of absolute staining intensities, a challenge that has also been highlighted as problematic in previous studies (e.g. Bollenbach et al. 2008, Gregor et al. 2007).

All data were derived from quantitative analysis of 50% epiboly embryos stained for pSmad2 as described in the Materials and methods section. Nuclear intensities from each embryo were normalized to the total gradient intensity (i.e., the integral of the activity gradient) to control for technical variability in antibody staining. A) Normalized Nodal activity gradients in wild type embryos. Twelve gradients derived from six embryos (i.e., gradients from the left and right sides of embryo cross-sections) are plotted in light blue. Each gradient is estimated with a sliding window average of average nuclear pSmad2 staining intensity. The mean gradient shape was calculated by taking a sliding window average of pooled data (dark blue). B) Normalized Nodal activity gradients in lft double mutants rescued by exposure to 2 µM SB-505124 starting at the 8-cell stage. Twelve gradients were derived from six embryos (light red), and the mean gradient was estimated with a sliding window average of pooled data (dark red). C) Mean wild type (blue) and rescued lefty double mutant (red) gradients are plotted for comparison. Error bars denote 1 standard deviation of data within 8 µm bins. Although the signaling gradient shapes differ between wild type and drug-rescued lft double mutant embryos, they appear to have similar variability. Note, however, that the accepted practice of normalizing by total intensity may diminish true biological variability in gradient amplitude.

3) One possible extension of the finding described is that the spatial requirement for Nodal signalling is not very stringent. Have the authors attempted a converse rescue experiment, i.e. to activate Nodal signaling uniformly, either with a drug or RNA injection in a cyclops;squint double mutant. Would they expect a just-right level of global signaling that rescues development?

It is well established that ectopic activation of Nodal signaling leads to ectopic mesendoderm induction in wild type embryos, but the reviewers are correct that it has not been tested stringently whether broad activation of the pathway can rescue Nodal signaling mutants. The genetics of creating cyc;sqt double mutants (1/16 embryos are double mutants) makes such an experiment extremely challenging. We have instead extended the experiments performed in Gritsman et al. 1999, in which the Nodal co-receptor mutant MZoep, which is completely devoid of Nodal signaling, is injected with activin mRNA. Activin bypasses the requirement for oep and is able to activate the Nodal signaling pathway in a dose-dependent manner (Dubrulle et al. 2015). We injected different amounts of activin mRNA into MZoep embryos at the one- to two-cell stage and observed partial rescue of some phenotypes, such as loss of trunk mesendoderm and, in one rare instance, rescue of cyclopia (new Figure 5—figure supplement 3). However, these embryos had severe morphological defects, in stark contrast to rescued lft double mutants (Figure 6D-F,H, Figure 6—figure supplements 1 and 2). We therefore conclude that the spatial requirements for Nodal activity are much stricter than for Lefty. We have updated the paper accordingly.

4) Regarding the statement "Thus, an ectopic, Nodal-independent source of Lefty at the animal pole can replace endogenous, Nodal-induced Lefty at the margin": Is this confirmed by fate-mapping (with Alexa 488-dextran) of the transplanted, Lefty-expressing cells? Figure 5E, F shows the position of cells shortly after transplantation, but it is quite possible that the transplanted cells move down into the margin during epiboly/gastrulation, providing a more localized source of exogenous Lefty.

We thank the reviewers for requesting this control experiment. Previous studies (e.g. Dubrulle et al. 2015, Chen et al. 2001, Müller et al. 2012) already showed that transplanted cells tend to remain at the animal pole. To directly determine the location of transplanted cells in our experiments, we injected Alexa 488-dextran, membrane RFP, and lft1 mRNA into lft double mutant embryos and transplanted cells from these donors into membrane RFP-expressing host lft double mutants at sphere stage, as in Figure 5. We then imaged host embryos for 4.5 hours post-transplantation, covering the stages during which the majority of germ layer patterning occurs (new Figure 5—figure supplement 1). We found that transplanted cells tended to remain at the animal pole and later frequently localized to the head, consistent with an animal pole localization at earlier stages (new Figure 5—figure supplement 2). Most importantly in the context of the paper, the transplanted cells represent a radically different spatial source of Lefty compared to the endogenous source at the margin.

5) It is somewhat surprising that there is not a graded effect of loss of lefty alleles, i.e. that the embryonic phenotypes and response readouts (in situs and pSmad2) do not show a graded response, and that loss of one, two or three alleles of Lefty1/2 seem to be equivalent. Is it correct that there is no dose response? What are the phenotypes of the genetic combinations with just one functional allele?

We thank the reviewers for pointing out this interesting observation. Embryos with even a single functional lft allele appeared largely morphologically normal (Figure 1Q, R), and gave rise to fertile adult fish, which we incrossed to obtain double homozygous embryos for several of the experiments in this study. These results are consistent with the feedback induction of lfts by Nodal signaling and the previous observation that lfts are highly sensitive Nodal target genes – they have the highest transcription rates of all targets in response to Nodal signaling, and lft transcripts are among the least stable (Dubrulle et al. 2015). Moreover, we also observed high sensitivity of lft2 expression to alterations in Nodal levels (Figure 7). We therefore speculate that robustness to lft allele dosage is the result of compensation from the remaining alleles, which may be induced rapidly in response to slightly increased Nodal signaling. We note, however, that subtler patterning defects may occur in lft-reduced embryos; for example, we did observe heart patterning defects in some lft1-/- mutants (Figure 1—figure supplement 1). In the future it will be interesting to more closely assess the patterning of Nodal-dependent structures in both lft1 and lft2 mutants in greater detail (e.g. lateral plate mesoderm, heart, endoderm, left-right positioning of organs, etc.).

6) In the Discussion, "While Lefty is not essential for viability per se" is an overstatement. The lefty double mutants are not viable. Although they can be experimentally rescued by drug or resupply of Lefty, this does not alter the requirement for functional Lefty during development (whether provided by expression or downstream pathway inhibition), as demonstrated by the double mutant phenotype. It is important in the interpretation of the results and the discussion of the results to uncouple the concept of dynamic feedback inhibition with the concept of the requirement of Lefty to be in the game, at least to modulate Nodal activity levels for viability.

We agree with the reviewers and removed this statement to change the sentence to: “The ability to dynamically adjust pathway activity may allow the embryo to create reliable patterns in the face of signaling fluctuations and uncertain environmental conditions”.

Contribution

Competing interests

Steven Zimmerman

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

Contribution

Resources, Methodology

Competing interests

No competing interests declared

Deniz C Aksel

Program in Biophysics, Harvard Medical School, Boston, United States

Contribution

Investigation

Competing interests

No competing interests declared

Deepak Reyon

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, United States

Department of Pathology, Harvard Medical School, Boston, United States

Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, United States

Center for Cancer Research, Massachusetts General Hospital, Charlestown, United States

Present address

Editas Medicine, Inc., Cambridge, United States

Contribution

Resources, Methodology

Competing interests

No competing interests declared

Shengdar Q Tsai

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, United States

Department of Pathology, Harvard Medical School, Boston, United States

Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, United States

Center for Cancer Research, Massachusetts General Hospital, Charlestown, United States

Present address

St. Jude Children’s Research Hospital, Memphis, United States

Contribution

Resources, Methodology

Competing interests

No competing interests declared

J Keith Joung

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, United States

Department of Pathology, Harvard Medical School, Boston, United States

Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, United States

Center for Cancer Research, Massachusetts General Hospital, Charlestown, United States

Contribution

Resources, Methodology

Competing interests

JKJ is a consultant for Horizon Discovery. JKJ has financial interests in Beacon Genomics, Editas Medicine, Poseida Therapeutics, and Transposagen Biopharmaceuticals. JKJ's interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Alexander F Schier

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

Broad Institute of MIT and Harvard University, Cambridge, United States

Center for Brain Science, Harvard University, Cambridge, United States

Arnold and Mabel Beckman Foundation (Postdoctoral Fellowship)

American Cancer Society (Postdoctoral Fellowship)

National Institutes of Health (AP-K99 -HD076935)

National Institutes of Health (JKJ-R01-GM088040)

National Institutes of Health (JKJ-DP1-GM105378)

J Keith Joung

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank María Almuedo-Castillo, Jeffrey Farrell, Hans Meinhardt, and Patrick Müller for insightful comments and discussions, Laila Akhmetova and Julien Dubrulle for experimental support, Antonius van Boxtel, John Chesebro, and Caroline Hill for sharing their pSmad2 immunofluorescence protocol, and Joseph Zinski and Mary Mullins for immunofluorescence imaging advice. Funding was provided by the National Science Foundation (KWR), the Arnold and Mabel Beckman Foundation (NDL), the American Cancer Society (JAG), and the National Institutes of Health (AFS GM056211, AP K99 HD076935). JKJ was supported by an NIH Director's Pioneer Award (DP1 GM105378) and NIH R01 GM088040. JKJ is a consultant for Horizon Discovery. JKJ has financial interests in Beacon Genomics, Editas Medicine, Poseida Therapeutics, and Transposagen Biopharmaceuticals. JKJ’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#25-08) of Harvard University.

Reviewing Editor

Deborah Yelon, Reviewing Editor, University of California, San Diego, United States

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